Herein we provide a detailed molecular analysis of the spatial heterogeneity of clinically localized, multifocal prostate cancer to delineate new oncogenes or tumor suppressors. We initially determined the copy number aberration (CNA) profiles of 74 patients with index tumors of Gleason score 7. Of these, 5 patients were subjected to whole-genome sequencing using DNA quantities achievable in diagnostic biopsies, with detailed spatial sampling of 23 distinct tumor regions to assess intraprostatic heterogeneity in focal genomics. Multifocal tumors are highly heterogeneous for single-nucleotide variants (SNVs), CNAs and genomic rearrangements. We identified and validated a new recurrent amplification of MYCL, which is associated with TP53 deletion and unique profiles of DNA damage and transcriptional dysregulation. Moreover, we demonstrate divergent tumor evolution in multifocal cancer and, in some cases, tumors of independent clonal origin. These data represent the first systematic relation of intraprostatic genomic heterogeneity to predicted clinical outcome and inform the development of novel biomarkers that reflect individual prognosis.

Rationale: The mechanisms underlying cystic fibrosis (CF) lung disease pathogenesis are unknown.Objectives: To establish mechanisms linking anion transport with the functional microanatomy, we evaluated normal and CF piglet trachea as well as adult swine trachea in the presence of selective anion inhibitors.Methods: We investigated airway functional microanatomy using microoptical coherence tomography, a new imaging modality that concurrently quantifies multiple functional parameters of airway epithelium in a colocalized fashion.Measurements and Main Results: Tracheal explants from wild-type swine demonstrated a direct link between periciliary liquid (PCL) hydration and mucociliary transport (MCT) rates, a relationship frequently invoked but never experimentally confirmed. However, in CF airways this relationship was completely disrupted, with greater PCL depths associated with slowest transport rates. This disrupted relationship was recapitulated by selectively inhibiting bicarbonate transport in vitro and ex vivo. CF mucus exhibited increased viscosity in situ due to the absence of bicarbonate transport, explaining defective MCT that occurs even in the presence of adequate PCL hydration.Conclusions: An inherent defect in CF airway surface liquid contributes to delayed MCT beyond that caused by airway dehydration alone and identifies a fundamental mechanism underlying the pathogenesis of CF lung disease in the absence of antecedent infection or inflammation.

The c-myc proto-oncogene encodes a transcription factor, c-Myc, which is deregulated and/or overexpressed in many human cancers. Despite c-Myc's importance, the identity of Myc-regulated genes and the mechanism by which Myc regulates these genes remain unclear. By combining chromatin immunoprecipitation with CpG island arrays, we identified 177 human genomic loci that are bound by Myc in vivo. Analyzing a cohort of known and novel Myc target genes showed that Myc-associated protein X, Max, also bound to these regulatory regions. Indeed, Max is bound to these loci in the presence or absence of Myc. The Myc:Max interaction is essential for Myc-dependent transcriptional activation; however, we show that Max bound targets also include Myc-repressed genes. Moreover, we show that the interaction between Myc and Max is essential for gene repression to occur. Taken together, the identification and analysis of Myc bound target genes supports a model whereby Max plays an essential and universal role in the mechanism of Myc-dependent transcriptional regulation.

No abstract

Noncompressible torso hemorrhage is a leading cause of death in trauma, with many patients dying before definitive hemorrhage control. Resuscitative endovascular balloon occlusion of the aorta (REBOA) is an adjunct than can be used to expand the window of salvage in patients with end-stage hemorrhagic shock. The aim of this study was to evaluate the effect of continuous and intermittent REBOA (iREBOA) on mortality using a highly lethal porcine model of noncompressible torso hemorrhage. Male splenectomized pigs (70-90 kg) underwent a laparoscopic liver injury (80% resection of left lobe) followed by a 10-min free-bleed period. Animals were then divided into three groups (n = 8) for a 60-min intervention phase (n = 8): continuous occlusion (cREBOA), iREBOA, or no occlusion (nREBOA). Groups then underwent whole blood resuscitation, damage control surgery, and further critical care. Endpoints were mortality and hemodynamic and circulating measures of shock and resuscitation. Systolic blood pressure (in mmHg) at the end of the free-bleed period for cREBOA, iREBOA, and nREBOA was 31 ± 14, 48 ± 28, and 28 ± 17, respectively (P = 0.125). Following the start of the intervention phase, systolic blood pressure was higher in the iREBOA and cREBOA groups compared with the nREBOA (85 ± 37 and 96 ± 20 vs. 42 ± 4; P < 0.001). Overall mortality for the cREBOA, iREBOA, and nREBOA groups was 25.0%, 37.5%, and 100.0% (P = 0.001). Resuscitative endovascular balloon occlusion of the aorta can temporize exsanguinating hemorrhage and restore life-sustaining perfusion, bridging critical physiology to definitive hemorrhage control. Prospective observational studies of REBOA as a hemorrhage control adjunct should be undertaken in appropriate groups of human trauma patients.

An effective tool for the global analysis of both DNA methylation status and protein–chromatin interactions is a microarray constructed with sequences containing regulatory elements. One type of array suited for this purpose takes advantage of the strong association between CpG Islands (CGIs) and gene regulatory regions. We have obtained 20 736 clones from a CGI Library and used these to construct CGI arrays. The utility of this library requires proper annotation and assessment of the clones, including CpG content, genomic origin and proximity to neighboring genes. Alignment of clone sequences to the human genome (UCSC hg17) identified 9595 distinct genomic loci; 64% were defined by a single clone while the remaining 36% were represented by multiple, redundant clones. Approximately 68% of the loci were located near a transcription start site. The distribution of these loci covered all 23 chromosomes, with 63% overlapping a bioinformatically identified CGI. The high representation of genomic CGI in this rich collection of clones supports the utilization of microarrays produced with this library for the study of global epigenetic mechanisms and protein–chromatin interactions. A browsable database is available on-line to facilitate exploration of the CGIs in this library and their association with annotated genes or promoter elements.

The c-myc proto-oncogene can direct a diverse array of biological activities, including cell cycle progression, apoptosis, and differentiation. It is believed that Myc can affect this wide variety of activities by functioning as a regulator of gene transcription, although few targets have been identified to date. To delineate the molecular program regulated downstream of Myc, we used a cDNA microarray approach and identified 52 putative targets out of >6000 cDNAs analyzed. To further distinguish the subset of genes whose regulation was dependent upon Myc per se from those regulated in response to activation of general mitogenic or apoptotic programs, the putative cDNA targets were then screened by a series of assays. By this approach 37 putative targets were ruled out and 15 Myc target genes were uncovered. Interestingly, comparing our results with other high throughput screens reveals that certain putative Myc targets previously reported are shown not to be regulated downstream of Myc (e.g. ribosomal proteins, HSP90␤), whereas others are further supported by our analyses (e.g. pdgf␤r, nucleolin). The identity of genes specifically regulated downstream of Myc provides the critical tools required to understand the role Myc holds in the transformation process and to delineate how Myc functions as a regulator of gene transcription.

Nuclear mutations are well known to drive tumor incidence, aggression and response to therapy. By contrast, the frequency and roles of mutations in the maternally inherited mitochondrial genome are poorly understood. Here we sequence the mitochondrial genomes of 384 localized prostate cancer patients, and identify a median of one mitochondrial single-nucleotide variant (mtSNV) per patient. Some of these mtSNVs occur in recurrent mutational hotspots and associate with aggressive disease. Younger patients have fewer mtSNVs than those who diagnosed at an older age. We demonstrate strong links between mitochondrial and nuclear mutational profiles, with co-occurrence between specific mutations. For example, certain control region mtSNVs co-occur with gain of the MYC oncogene, and these mutations are jointly associated with patient survival. These data demonstrate frequent mitochondrial mutation in prostate cancer, and suggest interplay between nuclear and mitochondrial mutational profiles in prostate cancer.